1. Field of the Invention
The present invention relates generally to cancer diagnostics and more specifically to methods for revealing circulating tumor cells (CTCs) allowing for robust clinical analysis of low volume samples.
2. Background Information
Circulating tumor cells (CTCs) are generally, although not exclusively, epithelial cells that originate from a solid tumor in very low concentration into the blood stream of patients with various types of cancer. The shedding of CTCs by an existing tumor or metastasis often results in formation of secondary tumors (see FIG. 1). Secondary tumors typically go undetected and lead to 90% of all cancer deaths. Circulating tumor cells provide the link between the primary and metastatic tumors. This leads to the promise of using the identification and characterization of circulating tumor cells for the early detection and treatment management of metastatic epithelial malignancies. Detection of CTCs in cancer patients offers an effective tool in early diagnosis of primary or secondary cancer growth and determining the prognosis of cancer patients undergoing cancer treatment because number and characterization of CTCs present in the blood of such patients has been correlated with overall prognosis and response to therapy (see FIGS. 2 and 3). Accordingly, CTCs serve as an early indicator of tumor expansion or metastasis before the appearance of clinical symptoms.
While the detection of circulating tumor cells (CTCs) has important prognostic and potential therapeutic implications in the management and treatment of cancer, because of their occult nature in the bloodstream, these rare cells are not easily detected. CTCs were first described in the 1800s, however only recent technological advances have allowed their reliable detection. CTCs are thought to exist in peripheral blood at ultra-low concentrations of patients with tumors. For example, for patients with carcinomas it is estimated that every one in ten million normal blood cells is a CTC. While existing technology can identify CTCs and correlate them with disease, no method has sufficient sensitivity to reliably measure a statistically significant number of cells at varying stages of the disease to guide the most effective treatment regime.
The first automated system developed to enumerate CTCs, was put on the market in 2004 and uses immunomagnetic enrichment technology. More recent methodologies have challenged this system as the “gold standard” for enumeration of CTCs, finding a factor of 10-100 times more CTCs. The reason(s) behind this apparent discrepancy in the numbers of CTCs found per blood sample with different methodologies is not understood, but has raised questions and concerns within the community of researchers and clinicians in this established field.
The most used methods for enumeration/characterization of CTCs are immunomagnetic enrichment methods targeting the surface protein EpCam, fiber-optic array technology, and a recently developed “CTC chip”.
With the use of immunomagnetic enrichment technology CTCs can be detected in between 45% and 50% of metastatic breast cancer patients. The most widely used methodology to detect CTCs utilizes immunomagnetic enrichment. The technology relies upon immunomagnetic enrichment of tumor cell populations using magnetic ferrofluids linked to an antibody which binds epithelial cell adhesion molecule (EpCAM), expressed only on epithelial derived cells. This methodology requires 7.5 mL of blood for analysis and finds greater than 2 CTCs in only some metastatic cancer patients as shown in the Table 1 below. Other studies have been done using negative enrichment, and other forms of positive enrichment, and have achieved similar results.
TABLE 1Detection of CTCs in Various Types ofCancers Using Immunomagnetic EnrichmentTypes of Cancers≧2 CTCs foundAve/7.5 mLAve/mlBreast (n = 422)37%8411Lung (n = 99)20%304Colorectal (n = 196)30%40.5Pancreatic (n = 16)19%20.3Prostate (n = 123)57%7510HD (n = 345) 1%0.10.01
Another method for enumeration/characterization of CTCs is Fiber-optic Array Scanning Technology (FAST). Using the FAST method, 7.5 mL of blood is needed for analysis. Red blood cells are lysed and nucleated cells are distributed as a monolayer on slides that can hold up to 30 million cells. There is no enrichment step in this methodology. Cells are fixed, permeabilized and stained with a pan anti-cytokeratin antibody-Alexa Fluor 555, CD45-Alexa Fluor 647, and DAPI (nuclear stain). FAST scans each slide and identifies the location of each red fluorescent object on the slide. Each fluorescent object is imaged via an automated digital microscope and CTCs are enumerated as being CK+, CD45−, DAPI+ cells. This methodology has been tested on a variety of metastatic cancer patients, including breast, lung, prostate, colorectal, and pancreatic. Similar CTC counts are found using this method as compared to methods using immunomagnetic enrichment.
Another method for enumeration/characterization of CTCs is microfluidic or “CTC-Chip” technology. The methods utilizes 1-3 mL of blood in which whole blood flows past 78,000 EpCam-coated microposts. EpCam+ cells stick to the posts and are subsequently stained with cytokeratin, CD45, and DAPI. With this methodology, CTCs are found in virtually all metastatic cancer patients at a relatively high purity and not in healthy controls. Additionally, CTC-chip technology identifies CTCs in all patients and in higher numbers than other technologies by a′factor of approximately 10 to 100 fold as reported in two recent publications shown in Tables 2 and 3 below.
TABLE 2Identification of CTCs Using CTC-Chip TechnologyTypes of CancersRange/mLAve/mLAve Purity*Breast (n = 10) 5 to 1767960%NSCLC (n = 55)  5 to 1,28115552%Prostate (n = 26)16 to 2928649%Pancreatic (n = 15) 9 to 83119653%Colorectal (n = 10)42 to 37512167%HD (n = 20)0n/an/a*Purity is the ratio between cells attached to the EpCam posts that are CK+ versus CD45+
TABLE 3Identification of CTCs Using CTC-Chip TechnologyType of CancerRange/mLAve/mLAve PurityNon-small cell lung carcinoma NSCLC5 to 77174not(n = 23)reported
Although all of the CTC detection approaches are currently in use, significant limitations have been identified with the current approaches. One limitation is that the number of CTCs detected per sample using current methods is too low to provide robust interpretation or clinically meaningful content of a particular sample. Additional limitations of current methods include low CTC detection due to CTC heterogeneity. For example, differences in individual CTC features within the CTC population of interest further hinder the number of CTCs detected using current methodologies. Such differences may include size variations between individual CTCs, and variable or down regulated expression between individual CTCs of the cell surface markers used to detect CTCs.